Pathomechanism of testicular inflammation in rat involves activation of proteinase activated receptor 2

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I O S I O A N R A D U U B P O M E H M E I N O L A T H C A N I S O F A O V V E S P A R

VVB LAUFERSWEILER VERLAG

édition scientifique

9 7 8 3 8 3 5 9 5 0 5 4 2

ISBN 3-8359-5054-1

VVB LAUFERSWEILER VERLAG S T A U F E N B E R G R I N G 1 5 D - 3 5 3 9 6 G I E S S E N Tel: 0641-5599888 Fax: -5599890 r e d a k t i o n @ d o k t o r v e r l a g . d e w w w . d o k t o r v e r l a g . d e

INAUGURALDISSERTATION

zur Erlangung des Grades eines

Doktors der Humanbiologie

des Fachbereichs Medizin der

Justus-Liebig-Universität Giessen

I O S I O A N R A D U U B P O M E H M E I N O L A T H C A N I S O F A O V V E S P A R

VVB LAUFERSWEILER VERLAG

édition scientifique

9 7 8 3 8 3 5 9 5 0 5 4 2

ISBN 3-8359-5054-1

VVB LAUFERSWEILER VERLAG S T A U F E N B E R G R I N G 1 5 D - 3 5 3 9 6 G I E S S E N Tel: 0641-5599888 Fax: -5599890 r e d a k t i o n @ d o k t o r v e r l a g . d e w w w . d o k t o r v e r l a g . d e

INAUGURALDISSERTATION

zur Erlangung des Grades eines

Doktors der Humanbiologie

des Fachbereichs Medizin der

Justus-Liebig-Universität Giessen

INFLAMMATION IN RAT INVOLVES

ACTIVATION OF PROTEINASE

ACTIVATED RECEPTOR 2

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Pathomechanism of Testicular Inflammation in Rat

Involves Activation of

Proteinase Activated Receptor 2

INAUGURALDISSERTATION zur Erlangung des Grades eines

Doktors der Humanbiologie des Fachbereichs Medizin der Justus-Liebig-Universität Giessen

vorgelegt von IOAN RADU IOSUB aus Piatra Neamt, Rumänien

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Aus dem Institut für Anatomie und Zellbiologie

Geschäftsführende Direktorin: Frau Prof. Dr. E. Baumgart-Vogt des Fachbereichs Medizin der Justus-Liebig-Universität Giessen

Gutachter: Prof. Dr. A. Meinhardt Gutachter: PD Dr. T. Monsees

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CONTENTS

CONTENTS ---I

LIST OF ABBREVIATIONS --- 1

1 INTRODUCTION--- 3

1.1 MORPHOLOGY OF THE TESTIS---3

1.1.1 Functional organization and hormonal regulation of the testis --- 3

1.1.2 The tubular compartment and spermatogenesis--- 4

1.1.3 The interstitial compartment--- 6

1.1.4 The testicular immune response --- 9

2.2 MALE INFERTILITY--- 12

1.3MODELS OF IMMUNOLOGICAL INFERTILITY--- 12

1.4 PROTEINASE-ACTIVATED RECEPTORS--- 13

1.4.3 PAR2 in inflammation---16

1.4.4 PAR2 in the testes---17

1.5AIM OF THE PROJECT--- 17

3 MATERIALS AND METHODS ---18

3.1. MATERIALS--- 18

3.1.1 Buffers and solutions---18

3.1.2 Oligonucleotides---19

3.1.3 Animals --- 21

3.2 METHODS--- 21

3.2.1 Induction of experimental autoimmune orchitis (EAO) ---21

3.2.2 PAR2 activation in vivo by human ß tryptase ---22

3.2.3 PAR2 activation in vivo using specific stable synthetic peptides --- 22

3.2.4 Myeloperoxydase (MPO) staining ---23

3.2.5 Staining and quantification of mast cells---23

3.2.6 PAR2 immunohistochemistry on testicular paraffin sections ---24

3.2.7 PAR2 immunohistochemistry on isolated peritubular cells ---25

3.2.8 Double immunofluorescence on isolated testicular macrophages ---25

3.2.9 Double immunofluorescence on testicular cryosections ---26

3.2.10 Cell culture experiments ---27

3.2.11 Assessment of intracellular free Ca2+ concentration ---29

3.2.12 Western Blot analysis ---30

3.2.13 Working with RNA ---31

3.2.14 Isolation of total RNA from cultured cells and tissues ---31

3.2.15 Assessment on RNA concentrations by spectrophotometric analysis ---32

3.2.16 DNase Digestion ---33

3.2.17 Reverse transcription---33

3.2.18 Polymerase chain reactions (PCR)--- 34

3.2.19 DNA agarose gel electrophoresis---35

3.2.20 Optimization of PCR reactions for real-time PCR ---36

3.2.21 Quantitative real time PCR---36

3.2.22 Statistics---38

4. RESULTS ---39

4.1. TESTICULAR ATROPHY AND DECREASED TESTICULAR WEIGHT ASSOCIATED WITH EAO ---- 39

4.2. INCREASED NUMBERS OF NEUTROPHILS IN EAO --- 40

4.3. STRONG INCREASE IN MAST CELL NUMBERS IN EAO--- 41

4.4. PAR2 IMMUNOREACTIVITY IS ELEVATED IN CHRONICALLY INFLAMED RAT TESTIS--- 43

4.5 DETECTION OF PAR2IN ISOLATED TESTICULAR MACROPHAGES--- 44

4.6 COMPLETE DIGESTION OF GENOMIC DNA IN TOTAL RNA SAMPLES--- 44

4.7 SUCCESSFUL REVERSE TRANSCRIPTION WAS PROVED BY GAPDH-PCR--- 45

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4.9 EXPRESSION PROFILES OF INFLAMMATORY MEDIATORS IN TESTICULAR AND PERITONEAL

MACROPHAGES STIMULATED WITH A PAR2 AGONIST--- 50

4.10 ISOLATED PERITUBULAR CELLS EXPRESS FUNCTIONAL PAR2 AND ACTIVATE SECOND MESSENGERS--- 53

4.11 PAR2 POSITIVE CELLS IN THE GRANULOMA EXPRESS SMOOTH MUSCLE ACTIN AND PROLIFERATE--- 57

4.12 EXPRESSION PROFILES OF INFLAMMATORY MEDIATORS IN EAO AND ISOLATED PERITUBULAR CELLS STIMULATED WITH A PAR2 AGONIST--- 59

4.13 IN VIVO ACTIVATION OF PAR2 IN RAT TESTIS--- 63

4.14 IN VIVO SIRNA GENE SILENCING IN RAT TESTIS--- 66

5. DISCUSSION ---70

5.1PAR2 IN THE NORMAL AND INFLAMED TESTIS--- 70

5.2 ROLE OF MAST CELLS IN TESTICULAR INFLAMMATION--- 72

5.3 PAR2 MEDIATED ACTION ON PTC--- 74

5.4MAST CELL TRYPTASE-PAR2 PATHWAY RESPONSIBLE FOR UPREGULATION OF KEY INFLAMMATORY MEDIATORS--- 75

5.5 PAR2 DEPENDENT MCP-1 UPREGULATION REQUIRES COX2 AND NO PRODUCTION. --- 77

5.6 MAST CELL TRYPTASE-PAR2 PATHWAY ON TESTICULAR MACROPHAGES--- 79

5.7IN VIVO GENE SILENCING BY LOCAL DELIVERY OF SIRNA INTO THE TESTIS--- 81

5.8 CONCLUSIONS--- 83 6 SUMMARY---86 7 ZUSAMMENFASSUNG ---88 8. REFERENCES---91 9. ACKNOWLEDGEMENTS--- 102 10. CURRICULUM VITAE --- 103 11. EHRENWÖRTLICHE ERKLÄRUNG --- 105

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LIST OF ABBREVIATIONS

bp Base pair

b.w. Body weight

BSA Bovive serum albumin cDNA Complementary DNA COX-2 Cyclooxygenase-2

DAPI 4’, 6’-diamidino-2-phenylindole, dihydrochloride DMEM Dulbecco’s Minimal Essential Medium

DMSO Dimethyl-sulfoxid DNA Deoxyribonucleic acid DNase Deoxyribonuclease

dNTPs 2’-deoxynucleoside-5’-triphosphates EAO Experimental autoimmune orchitis EDTA Ethylene diaminetetraacetic acid FCS Fetal calf serum

GAPDH Glycerin-Aldehyd-Phosphat-Dehydrogenase

HEPES (2-Hydroxyethyl)-1-piperazineethanesulphonic acid HRP Horse radish peroxidase

iNOS Inducible nitric oxide synthase

kD Kilodalton

LPS Lipo-polysacharide

MCP-1 Monocyte chemoattractant protein-1 MPO Myeloperoxidase

NaCl Sodium Chloride

PAR2 Proteinase activated receptor-2

PAR2-AP PAR2 activating peptide (SLIGRL-NH2)

PAR2-f-AP PAR2 modified activating peptide ([2-furoyl]-LIGRLO-NH2)

PAR2-RP PAR2 reverse peptide (LSIGRL-NH2)

PBS Phosphate buffered saline PCR Polymerase chain reaction RNA Ribonucleic acid

RNase Ribonuclease

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RT-PCR Reverse transcription SDS Sodium-dodecyl- sulphate sma Smooth muscle actin TAE Tris-acetate-EDTA buffer

TE Tris-EDTA

TGFß-2 Transforming growth factor-betta-2 Tris Tris(hydroxymethyl)-amino-methane

U Unit

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1

INTRODUCTION

1.1 Morphology of the testis

The male reproductive system has to fulfill three major functions: steroidogenesis (production of male sex steroid hormones), spermatogenesis (production of the male gametes) and delivery of the male gametes to the female reproductive tract. The testes are anatomically and functionally compartmentalized to accomplish the first two of these tasks. Histologically the organ is separated into an endocrine (interstitial) and a gametogenic (tubular) compartment. The male gonad is contained in a tough fibrous capsule called tunica albuginea. In the human, the testis is partitioned by connective tissue septa into discrete lobules containing the loops of the seminiferous tubules, which are connected at both ends to a reservoir termed rete testis located along one pole of the testis in the mediastinum. In contrast, rodent species such as rat and mouse display only free intertubular connective tissue with no distinct septa separating the seminiferous tubules (Huckins and Clermont, 1968, Fawcett et al., 1973)

1.1.1 Functional organization and hormonal regulation of the testis The interstitial compartment completely surrounds the seminiferous tubules and contains the androgen producing Leydig cells. Moreover, in the interstitial tissue the vasculature, lymphatic vessels and nerves of the testis are found. The seminiferous tubules are the gamete forming compartment of the testis. The seminiferous tubules are covered by a circumferential layer mainly consisting of peritubular myoid cells and the acellular components of the basal lamina (basement membrane), which together form the limiting tissue to the interstitial compartment on which the Sertoli cells and the most basally located spermatogenic cells rest.

Male reproduction is regulated and maintained by pulsatile secretion of gonadotropin releasing hormone (GnRH) by the hypothalamus under the control of the central nervous system. GnRH stimulates concordant pulses of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary (Fig. 1.1.1) (Leung and Steele, 1992, Huhtaniemi, 1995). LH

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stimulates Leydig cell development, morphology and secretion of androgens, mainly testosterone (Coquelin and Desjardins, 1982, Ellis and Desjardins, 1982, Sisk and Desjardins, 1986). Testosterone and FSH bind to specific receptors on Sertoli cells to regulate spermatogenesis and Sertoli cell functions directly such as secretion of inhibin. Spermatogenic cells do not respond to testosterone and FSH as they do not express the respective receptors. In turn, androgens and inhibin exert a negative feedback loop at the pituitary and hypothalamic level to regulate LH and FSH production (Coquelin and Desjardins, 1982, Ellis and Desjardins, 1982, Leung and Steele, 1992).

Fig. 1.1.1 Hormonal regulation of the testis (Gartner L. P., 2001) 1.1.2 The tubular compartment and spermatogenesis

The Sertoli cells provide the structural framework for the organization of the seminiferous epithelium, but also play a crucial role in supporting and directing the development of the spermatogenic cells. These cells remain in

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intimate contact with their adjacent Sertoli cells at all times during the spermatogenetic process, with junctional and membrane specializations providing physical contact and communication and forming the so called “blood-testis barrier” (Cheng and Mruk, 2002).

The spermatogenic cells start out as mitotically dividing precursors called spermatogonia, sitting on the basal lamina (Fig. 1.1.2). At puberty these mitotically dividing cells enter into meiosis at regular intervals, moving from the periphery of the tubule to the luminal part and becoming primary and later secondary spermatocytes. Meiosis leads to chromosomal rearrangements leading to production of haploid round spermatids (early spermatids), that subsequently undergo structural differentiation to become mature or elongated spermatids (late spermatids). Once these cells are released by the Sertoli cell into the lumen of the tubule they are called spermatozoa and the fluid secreted by the Sertoli cells sweeps the released immotile spermatozoa to the rete testes.

Fig. 1.1.2 Histology of the testis (J. W. Heath, 2000) A) longitudinal section through the testis stained with hematoxilin-eosin (HE) E-epididimis, RT-rete testis, TA-tunica albuginea; B) section through testicular parenchima HE-stained; C) HE staining of a cross-sectioned seminiferous tubule PC-peritubular cell, SC- Sertoli cell nucleus, SG-spermatogonia, 1Sc-primary spermatocyte, 2Sc-secondary spermatocytes, rS-round spermatids, eS-elongated spermatids.

Tight junctions between adjacent Sertoli cells and their associated membrane specializations form an intercellular barrier, which is completely impermeable for even small molecules (Whitehead, 1999, Cheng and Mruk, 2002). This blood-testis barrier separates the spermatogonia and early meiotic cells in the basal region of the seminiferous epithelium from the adluminal spermatocytes and spermatids. In this way a large majority of the developing germ cells are

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sequestered behind a physical barrier and effectively isolated from the immune system.

1.1.3 The interstitial compartment

The interstitium of the testis consists of connective tissue cells, steroidogenic Leydig cells, the vasculature and immune cells.

The vascular supply to the testis arises from the abdominal aorta and in species with scrotal testes this results in a comparatively long and highly coiled spermatic artery. The arterioles, capillaries and venules of the testis completely permeate the interstitial tissue. The venous drainage of the testis via the spermatic veins is very closely associated with the arterial supply and involves a very effective counter-current heat and solute exchange structure called the pampiniform plexus. The lymphatics of the testis are variable between species, from irregular channels incompletely bounded by endothelial cells in rodents, to large discrete lymphatic vessels in humans (Ghinea and Milgrom, 1995).

1.1.3.1 Leydig cells

Leydig cells are representing the majority of interstitial cells in the testis and are responsible for testosterone production. Various growth factors (interleukin 1alpha, transforming growth factor beta, inhibin, insulin-like growth factors I and II, vascular endothelial growth factor, and relaxin-like growth factor) modulate Leydig cell differentiation, regeneration, and steroidogenic capacity (Haider, 2004). Resident macrophages in the interstitial tissue of the testis are important for differentiation and endocrine function of Leydig cells (Hales, 2002). Acute testicular and systemic inflammation is accompanied by a decrease in the spermatogenic function of Leydig cells (Gow et al., 2001, Hedger et al., 2005) and the numbers of Leydig cells are considerably lower in EAO testis (Suescun et al., 1994, Suescun et al., 2001) probably due to apoptosis of Leydig cells which can be induced by cytotoxins.

1.1.3.2 Immune cells in the testis

The testis in spite of its immune privileged status is not isolated from the immune system (Hedger and Culler, 1997). Macrophages are observed in the interstitium of most species and many testes also contain variable numbers of

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eosinophils. In addition to the resident macrophages, mast cells are found adjacent to subcapsular blood vessels (Nistal et al., 1984, Gaytan et al., 1989). Less numerous, but ubiquitous are the intratesticular lymphocytes (Dym and Romrell, 1975). As in most other tissues, circulating immune cells, including T lymphocytes, also have relatively free access to the testis (Hedger and Meinhardt, 2000), and the testis has an efficient and effective lymphatic drainage to regional lymph nodes. Therefore, locally produced cytokines as well as those in the general circulation have the potential to exert effects at the testicular level.

The testicular macrophages

In rat and mouse the ratio of macrophages to Leydig cells is 1:4 (Hume et al., 1984). Macrophages display a close physical and functional relationship with the Leydig cells described as highly-specialized cytoplasmic interdigitations (Bambino and Hsueh, 1981).

The majority of testicular macrophages express a marker for tissue-resident macrophages (ED2, CD163). However, about 15-20% of the total testicular macrophages do not synthesize ED2. These cells can be identified by the expression of the lysosomal antigen CD68, recognized by antibody ED1 (Mahi-Brown et al., 1987, Wang et al., 1994, Gerdprasert et al., 2002a, Hedger, 2002). Moreover, 50% of the ED2+ cells are ED1- indicating the existence of several populations of macrophages in the rat testis, putatively representing different stages of development and/or functional stages (Hedger, 2002). ED1+ monocytes are recruited by chemokines into the testis where they become resident macrophages (ED2+) loosing the CD68 (ED1) marker. During this process, at a certain intermediate developmental step, these cells express both markers.

An increased number of ED1+ and ED2+ macrophages has been reported even at early stages of orchitis and more pronounced at later time points (Lustig et al., 1993).

Testicular dendritic cells

In spite of the common lineage, dendritic cells are morphologically distinct from macrophages and lack the phagocytic and killing capabilities of macrophages, whereas they are much more effective as antigen presenting

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cells (APCs) (Banchereau and Steinman, 1998). Dendritic cells have been observed in the testis of rat (Head and Billingham, 1985) mouse (Itoh et al., 1995, Hoek et al., 1997) and human (Becker et al., 1987, Derrick et al., 1993). The functional role of these cells in the testis remains unclear although it appears that they direct immune responses within the testis and adjacent lymph nodes.

Testicular lymphocytes

In rat and human testis, lymphocytes represent 10-20% of the total leukocyte population. T cells (CD4+ and CD8+) and natural killer (NK) cells comprise the specific subsets of lymphocytes that have been described in the normal rat, mouse and human testis (Ritchie et al., 1984, Pollanen and Niemi, 1987, Pollanen and Maddocks, 1988, Tompkins et al., 1998). The number of testicular lymphocytes are expanded in the testes of man with infertility and sperm autoimmunity. The T cells tend to recirculate throughout tissues where they initially encountered an antigen (Picker and Butcher, 1992), suggesting that expanded T cell populations in human infertile testes may be specific to testicular autoantigens.

Testicular mast cells

The distribution of granulocytes within the testis is species-specific. Mast cells are almost absent from the testicular interstitium of rat, mouse, cat, bull, dog and deer. They are associated with blood vessels at the periphery of the testes directly under the capsule. Contrary to the mentioned species, mast cells are found throughout the interstitial tissue in human testis (Nistal et al., 1984, Yamanaka et al., 2000a). It is most likely that mast cells play a role in local innate immunity. In humans, testicular mast cell numbers increase in various types of male infertility (Yamanaka et al., 2000a), but decline with increasing age (Nistal et al., 1984).

In the adult rat, mast cells proliferate dramatically throughout the testicular parenchyma following ablation of Leydig cells by EDS (Wang et al., 1994). The degree of proliferation appears to be under the control of Leydig cells, suggesting that these cells produce an inhibitor of the mast cell activity.

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Tryptase is found in mast cells of the human adult testis and is present in interstitial and peritubular mast cells, both in testes with normal spermatogenesis and in infertile testes. Mast cells are significantly increased in testes with spermatogenesis defects, suggesting that tryptase released from mast cells is a factor involved in male infertility (Meineke et al., 2000). Therefore tryptase appears as an interesting target in the treatment of male infertility.

Testicular neutrophils

In contrast to mast cells and eosinophils, neutrophils are absent from the interstitium of normal testis. They are found in the testis only in conditions of testicular inflammation or damage (Kohno et al., 1983, Gerdprasert et al., 2002a). They are chemoattracted by MCP-1, but it seems that higher levels of MCP-1 are required to chemoattract neutrophils, as compared to the levels required for the recruitment of macrophages.

1.1.4 The testicular immune response

Inflammation occurs in response to numerous stimuli and involves activation of monocytes, macrophages and mast cells, leading to production of pro-inflammatory cytokines, prostaglandins and cytotoxic reactive oxygen species, up-regulation of adhesion molecules, recruitment of immune cells, changes in blood flow and increased capillary permeability. If foreign antigen is present, an immunological response may also be triggered, involving antigen-specific T and B-lymphocytes, which also respond to specific cytokines.

Numerous clinical and experimental studies have shown that both local and systemic infection cause a transient down-regulation of androgen production, and disruption of germ cell production (O'Bryan et al., 2000). Several inflammatory mediators including cytokines are produced within the normal testis, where they are believed to be involved in regulating Leydig cell function and spermatogenic development (Hales et al., 1999, Hedger and Meinhardt, 2003). Production of these mediators is increased by inflammatory stimuli. The apparent overlap between testicular and immune regulatory mechanisms could provide the key to understanding both the processes leading to inflammation-mediated damage of testicular function and the phenomenon of

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immune privilege in the testis. Interestingly, the non-immune cells of the testis such as Sertoli, Leydig and peritubular myoid cells are found to contribute significantly, to the overall production of cytokines.

MCP-1 in the testis

MCP-1 belongs to a family of proteins called chemotactic cytokines which are important regulators of the leukocyte recruitment process and play an important role in inflammation (Kunkel and Butcher, 2002). Structurally, chemokines are proteins with low molecular mass characterized by four conserved cysteines forming two disulfide bridges. The position of the first two cysteines has been used to divide the chemokine family into two main subfamilies, the CXC chemokines and CC chemokines (Proost et al., 1996). MCP-1 is a member of the CC subfamily and is the chemokine that most potently acts on mononuclear cells, both monocytes and lymphocytes and on neutrophils (Proost et al., 1996).

It has been reported that in autoimmune diseases such as multiple sclerosis, arthritis and psoriasis, chemokines might be responsible for leukocyte migration to the inflamed tissue (Baggiolini and Dahinden, 1994) and for the local stimulation of leukocytes to release proteases. Concerning the testis, it was demonstrated in vitro production of MCP-1 by peritubular and Leydig cells where MCP-1 expression was markedly stimulated by some cytokines and LPS (Aubry et al., 2000). Recent studies (Gerdprasert et al., 2002a, Gerdprasert et al., 2002b) suggested that in a LPS inflammatory model the increase in the number of intratesticular macrophages was stimulated by MCP-1. Large amounts of MCP-1 are released during experimental autoimmune orchitis (Guazzone et al., 2003), but the underlying mechanisms are not elucidated.

TGFßs in the testis

The transforming growth factor-ß (TGFß) family members are dimeric cytokines with predominantly immunosuppressive and anti-inflammatory activities. TGFß is produced by macrophages and lymphocytes. In testis TGFß isoforms (1–3) are highly expressed by Sertoli cells, peritubular cells and Leydig cells in the foetal and immature testis, although production declines dramatically post-puberty (Mullaney and Skinner, 1993, Avallet et al.,

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1994). In addition, in the post-pubertal testis they also have been localised to the developing germ cells in a developmentally specific pattern of expression (Caussanel et al., 1997). The receptors for TGFß are found in both somatic and germ cells (Le Magueresse-Battistoni et al., 1995). Consequently, these cytokines have been involved in controlling both Leydig cell and seminiferous tubule development (Kohno et al., 1983). A precise role in the adult testis has yet to be established, although TGFß has been proposed to enhance immune privilege in the cryptorchid rat testis (Pollanen et al., 1993), and has been implicated in the immuno-protective activity of Sertoli cells in co-transplantation studies (Suarez-Pinzon et al., 2000).

Inducible nitric oxide synthase

NO is synthesized from L-arginine by the action of NO synthase (NOS), an enzyme existing in three isoforms. Brain NOS (bNOS) or neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3), also referred to as constitutive NOS (cNOS), are responsible for the continuous basal release of NO and both require calcium/calmodulin for activation (Griffith and Stuehr, 1995, Snyder, 1995). A third isoform is an inducible calcium-independent form (iNOS or NOS2) that is expressed only in response to inflammatory cytokines and lipopolysaccharides (LPS); reviewed in (Nussler and Billiar, 1993, Morris and Billiar, 1994). Several studies are reporting constitutive expression of iNOS in rat testes and increased iNOS levels associated with LPS induced acute systemic inflammation (O'Bryan et al., 2000, Gerdprasert et al., 2002a). More recent reports are showing a significantly statistical correlation between the iNOS immunoreactivity and mast cell numbers in the testis of man with impaired spermatogenesis (Sezer et al., 2005).

Cyclooxygenase 2

Cyclooxygenase 2 (COX-2) is constitutively expressed in the rat testis (Neeraja et al., 2003b) and overexpressed in testes of men with impaired spermatogenesis and in testicular cancer biopsies. Coculture studies of a human mast cell line and human orbital fibroblasts have indicated up-regulation of cyclooxygenase 2 in fibroblasts (Smith and Parikh, 1999) and productionof PGE2. Other reports indicated that IL-1ß (Inoue et al., 1997)or

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and prostaglandins synthesis. A recent study showed that mast cell tryptase, activating PAR2 is responsible for the induction of COX-2 expression in

human testis (Frungieri et al., 2002).

2.2 Male infertility

The autoimmunity in the form of antisperm antibodies and epididymo-orchitis is a significant cause of sub- and infertility in men. Inflammation due to reproductive tract infections, even systemic infection and illness, can lead to a failure of testicular androgen and sperm production (Bohring and Krause, 2003). The obvious capacity of the testis for inflammatory responses is contrasting with the fact that it is also one of a very few organs of the body capable of sustaining foreign grafts for extended periods without evidence of rejection (Head and Billingham, 1985). The so-called “immunological privilege” of the testis is believed to arise from the need to prevent immune responses against the auto-antigens of the meiotic and haploid germ cells, which first appear in the testis at the time of puberty, long after the establishment of self-tolerance in the perinatal period. The testis, in spite of its immune privileged status, is not isolated from the immune system (Hedger, 1997) having a population of resident macrophages as well as numerous mast cells adjacent to subcapsular blood vessels (Nistal et al., 1984, Anton et al., 1998). A variety of acute or chronic animal models were used to mimic the immunological male factor infertility, and many of them brought important contribution in understanding the underlying pathophysiological mechanisms.

1.3 Models of immunological infertility

Experimental autoimmune orchitis

Acute or chronic inflammation of the male genital tract may result in alterations of spermatogenesis, steroidogenesis and fertility (Bohring and Krause, 2003). Chronic orchitis in men usually occursas a consequence of different injuries induced by trauma or infectious agents. The interaction of immune cellswith spermatic antigens is one of the pathogenic mechanisms involved in testis autoimmunity. Different models of experimentalautoimmune orchitis (EAO) have been useful in understanding testicular cell interactions under pathological conditions. EAO has been induced in differentspecies by active immunization with spermatic antigens, by adoptiveT cell transfer or by

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neonatal thymectomy (Tung and Teuscher, 1995). EAO was induced in mouse (Kaneko et al., 2003, Watanabe et al., 2005) and rat (Doncel et al., 1989, Lustig et al., 1993, Guazzone et al., 2003), with most studies being performed in the rat EAO model.

Testicular damage resulting from EAO is characterized initially by a mild interstitial lymphomononuclear cell infiltrate and focal damage to the seminiferous epithelium. As the disease progresses, substantial interstitial immune cell infiltrates can be observed, spermatogenesis cessation, sloughing of the tubules and granuloma formation take place. The main target of the immunological attack of EAO are the germ cells that undergo apoptosis (Theas et al., 2003).

Acute orchitis

Acute testicular inflammation is studied in different experimental models, either as part of a systemic inflammatory process (Aubry et al., 2000, O'Bryan et al., 2000, Gerdprasert et al., 2002a, Gerdprasert et al., 2002b) or as isolated testicular inflammation induced by local traumatism or by testicular torsion (Ozturk et al., 2003). High levels of MCP-1 and iNOS, as well as increased numbers of ED1+ macrophages are reported in acute orchitis. In humans, the most frequent causes of acute testicular inflammation are mechanical injuries (trauma, testicular torsion) and mumps orchitis.

1.4 Proteinase-activated receptors

The protease-activated receptors (PARs) belong to a large superfamily of G-protein-coupled receptors and mediate the cellular actions of certain serine proteases. Four members, PARs 1 to 4, have been cloned within the past decade. PAR-1, -3 and -4 are alternative thrombin receptors with different tissue distributions; roles for these three distinct thrombin receptors in the activation of platelets have been well described. PAR2 is activated by trypsin,

mast cell tryptase and coagulation factors VIIa and Xa, but not by thrombin. These proteolytic enzymes cleave an N-terminal peptide at a specific site, and the newly exposed N-terminal end binds to the second extracellular loop of the receptor (Nystedt et al., 1994, Lerner et al., 1996, Al-Ani et al., 1999) (Fig. 1.4.2). Thus, the new receptor N-terminus functions as a ‘tethered ligand’ and

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activates the receptor, resulting in intracellular signaling mainly by Gq proteins (Vergnolle et al., 2001, Steinhoff et al., 2005).

Fig. 1.4.2 Enzymatic and non-enzymatic PAR2 activation, taken from (Kawabata, 2002). Enzymatic PAR2 activation

The N-terminal cleavage/activation site of PAR2 can be cleaved by trypsin,

mast cell tryptase and coagulation factors VIIa and Xa, yielding the new N-termini NH2-SLIGRL for murine PAR2 and NH2-SLIGKV for human PAR2. Tryptase is secreted by activated mast cells during inflammation and is a strong endogenous activator of human PAR2 (Molino et al., 1997, Compton et

al., 2001). Factors VIIa and Xa might become available as agonists for PAR2

expressed by the vascular endothelium in conditions such as disseminated intravascular coagulation (Camerer et al., 2000) and might also activate PAR2

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Non-enzymatic PAR2 activation

Non-enzymatic activation of PAR2 is achieved by synthetic peptides as short

as five or six amino acids, such as SLIGRL-NH2 and SLIGKV-NH2, based on

the receptor-activating sequence of the tethered ligands (Fig.2.4.2). These are capable of binding directly to the body of PAR2, mimicking the actions of the

endogenous activators. Recently a newly derived PAR2 activating peptide

2-Furoyl-LIGLRO-NH2 (PAR2-f-AP) was reported to have a markedly increased

activity, anenhanced selectivity for PAR2 and to show more stability in an in

vivo environment as compared to classical PAR2-AP. This compound should

prove to be very useful for studies done in intact animals that are aimed at elucidating the potential patho-physiological responses due to PAR2 when

activated invivo by locally generated proteinases (McGuire et al., 2004a).

Development of PAR2 antagonists would not only dramatically facilitate

understanding of the physiological and pathophysiological roles of PAR2, but

might also be valuable to the clinical treatment of certain human diseases. Activation of PAR2 induces assembly of a mitogen-activated protein kinases

(MAPK) signaling module activating the extracellular-signal-regulated kinases (ERK1/2). By the other hand PAR2 couples to Gßq/11, resulting in activation of

phospholipase C, production of inositol 1,4,5-trisphosphate and diacylglycerol, mobilization of Ca2+ and activation of protein kinase C. These signaling events are rapidly attenuated and desensitized after repeated stimulation, indicative of receptor desensitization and down-regulation. PAR2 activation induces

translocation of ß-arrestins to the plasma membrane, where they interact with PAR2 to mediate both desensitization and endocytosis (Dery et al., 1999,

DeFea et al., 2000). At one extreme, PARs, which are activated by irreversible proteolysis and are thus single-use receptors, traffic to lysosomes, a process termed down-regulation (Bohm et al., 1996). The down-regulation of receptors is of fundamental importance in terminating signaling. However, little is known about the molecular mechanisms of post-endocytic sorting that target receptors for degradation. Sustained signaling requires the mobilization of PAR2 from prominent stores in the Golgi apparatus or synthesis of intact

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1.4.3 PAR2 in inflammation

Numerous studies suggest that PAR2 contributes to the development of

inflammation. PAR2 activation leads to increased vascular permeability

(Kawabata et al., 1998, Vergnolle et al., 1999), blood vessel relaxation (Al-Ani et al., 1999, Sobey et al., 1999), systemic hypotension, bronchoconstriction (Ricciardolo et al., 2000), granulocyte infiltration (Vergnolle et al., 1998b) and leukocyte adhesion and margination (Vergnolle, 1999). These inflammatory responses, which can be induced by surgical exposure of tissues, are delayed in PAR2 deficient mice (Lindner et al., 2000), suggesting that PAR2 is an

important receptor in inflammation and is necessary for early inflammatory responses in vivo.

Neurogenic inflammation is a form of inflammation that depends on primary spinal afferent neurons with cell bodies in the dorsal root ganglia close to spinal cord and projections to the spinal cord and peripheral tissues (McDonald et al., 1996). Inflammatory agents trigger the release of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) from the peripheral endings of these neurons. SP interacts with the neurokinin 1 receptor (NK1R) on endothelial cells of post-capillary venules to cause plasma extravasation and granulocyte infiltration, and CGRP interact with its type 1 receptor on arteriolar smooth muscle to cause vasodilatation.

PAR2 agonists can also trigger processes that protect against inflammation.

PAR2 agonists improve cardiac function and reduce tissue damage after

myocardial ischemia (Napoli et al., 2000). PAR2 agonists can also serve to

protect the airway by acting as bronchodilators in certain species (Cocks et al., 1999). Systemic injection of PAR2 activating peptide to mice protects from

colitis and decreases pro-inflammatory cytokine synthesis and lethality (Fiorucci et al., 2001). PAR2 activation protects also the gastric mucosa

against the injurious effect of non-steroidal anti-inflammatory drugs (Kawabata et al., 2001). According the physiological or pathophysiological environments, PAR2 may be involved in different pro- or anti-inflammatory processes.

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1.4.4 PAR2 in the testes

Initially PAR2 expression hasbeen reported in cells of the testicular germinal

epithelium (D'Andrea et al., 1998). Later studies described PAR2

immunoreactivity on the acrosome of ejaculated spermtozoa (Weidinger et al., 2003) and in a fraction of the interstitial cells in the human testis. However, the precise nature of these PAR2-positive interstitial cells was not specified

(Frungieri et al., 2002). PAR2 activation by mast cell tryptase was found to be

responsible for the proliferation of peritubular cells in infertile testis and thus leading to the fibrotic thickening of the seminiferous tubules wall.

1.5 Aim of the project

Inflammation due to reproductive tract infections, even systemic infection and illness, can lead to a failure of testicular androgen and sperm production (Bohring and Krause, 2003). Immunological factor male infertility is one of the major causes of infertility in human and certain forms of male infertility have been found to be associated with increased numbers of tryptase containing testicular mast cells (Meineke et al., 2000). Moreover, recent advances are beginning to unravel the intriguing role mast cells play both in various autoimmune diseases (Benoist and Mathis, 2002, Robbie-Ryan and Brown, 2002) and in acute inflammation (Cocks and Moffatt, 2001, Roche et al., 2003b).

Proteinase-activated receptor-2 (PAR2), a G-protein coupled receptor

important to injury responses, was shown to be activated by mast cell tryptase. This study aimed to investigate the involvement of mast cells and PAR2 in the development and/or aggravation of acute and chronic testicular

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3

MATERIALS AND METHODS

3.1. Materials

3.1.1 Buffers and solutions

10X DNase I buffer:  0.1M TRIS (pH 8.3)

 0.5M KCl

 15mM MgCl2 6X DNA loading buffer:

 0.25% (w/v) bromophenol blue

 30% glycerol in H2O

HEPES buffer salt solution (1l, pH 7.4, sterile filtered)  418mg KCl  7.97 g NaCl  1ml MgCl2x6H2O  2.2ml CaCl2x2H2O  2.18g Glucose  2.38g HEPES Michaelis buffer (0.1M, 1l, pH 7.4):  20.6g sodiumbarbital  58.1ml HCl (1N)

50X TAE electrophoresis buffer (1l, pH 8.0):  242g of Tris base

 57.1ml of glacial acetic acid

 100ml of 0.5M EDTA

TE8:

 10 mM Tris (pH 8.0)

 1 mM EDTA (pH 8.0)

Toluidin blue (stock solution):

1% Toluidin blue O (Waldeck, Münster, Germany) (w/v) in 70% ethanol (v/v)

Toluidin blue (working solution): Dilute the stock solution 1:10 in freshly

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10X PBS (1l):  87.6g NaCl

 22.8g K2HPO4  6.8g KH2PO4

3.1.2 Oligonucleotides

All oligonucleotides were designed using Primer3 software available online at

http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi. Primer sequences, annealing temperatures and other technical details for PCR setup are presented in Table 3.1.2 All nucleotides were supplied by MWG-Biotech, Ebersberg, Germany. All primers were diluted at 100 pM/µl in 0.2x TE8 buffer and stored as stock solution at -20°C.

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3.1.3 Animals

Male inbred Wistar rats (Charles River, Sulzfeld, Germany) were used for all aspects of this study. Peritubular cells (PTC) were prepared from 19-day-old rats while for the in-vivo experiments and for the isolation of macrophages adult 200g rats were used. Experimental procedures were approved by the local authority (Regierungspraesidium Giessen) and conform to the Code of Practice for the Care and Use of Animals for Experimental Purposes.

3.2 Methods

3.2.1 Induction of experimental autoimmune orchitis (EAO)

Decapsulated testes from 200g rats were homogenized under sterile conditions at 4°C in an equal volume of isotonic saline buffer (500mg/ml wet weight) and stored at –20°C prior to use. Thirty rats 180-220g body weight (b.w.) were anaesthetized by intraperitoneal administration of 100mg/kg b.w. Ketamine (Ketavet, Pharmacia GmbH, Erlangen, Germany) and 10mg/kg b.w. Xylazine (Rompun, Bayer Vital GmbH, Leverkusen, Germany) and then immunized with 0.4ml syngenic testicular homogenate mixed with 0.4ml complete Freund’s adjuvant (Sigma-Aldrich, USA). Testes homogenates were mixed with the adjuvant solution few hours prior to immunizations. Three times at 14-day intervals, a total of 0.8ml/rat/timepoint was subcutaneously injected into the hind footpads and in different sites of the back skin. The wounds in the hind footpads were sealed with Histoacryl® tissue glue (Braun, Tuttlingen, Germany). The first two immunizations were followed by an intravenous injection (in the tail vein) of 1010 cells of inactivated Bordetella pertussis bacteria (DSMZ, Braunschweig, Germany) dispersed in 0.5ml isotonic saline, whilst the third was followed by intraperitoneal injection of 5x109 cells in 0.5ml isotonic saline (Doncel et al., 1989). Control animals (n=16) received complete Freund’s adjuvant containing Bordetella pertussis but no testis homogenate. Fifty and eighty days after the first immunization, the animals were sacrificed by overdose of Isofluran (FORENE®, Abbott, Wiesbaden, Germany). The testes were removed under sterile conditions, weighted and either snap frozen in liquid nitrogen or placed in Bouin’s solution for 12hrs. The fixed tissue was subsequently embedded in paraffin following standard procedures.

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3.2.2 PAR2 activation in vivo by human ß tryptase

Rats (200g; n=5-10/group) were anesthetized by intraperitoneal administration of 100mg/kg b.w. Ketamine (Ketavet, Pharmacia GmbH, Erlangen, Germany) and 10mg/kg b.w. Xylazine (Rompun, Bayer Vital GmbH, Leverkusen, Germany). The scrotum was carefully opened by a small incision.

Rats received by injection directly under the testicular capsule in both testes (using a thin insulin needle connected to a 1ml syringe) 50µl of a) 100nM recombinant ß tryptase diluted in isotonic saline containing 30µg/ml heparin (used to stabilize the tryptase), b) 100nM inhibited recombinant ß tryptase (with the irreversible inhibitor Pefabloc SC; residual tryptase activity ≤0.1%) diluted in isotonic saline containing 30µg/ml heparin; c) isotonic saline containing 30µg/ml heparin and d) isotonic saline alone (sham control). The injection site was always right of the testicular artery and special care was taken to minimize any damage to the testicular tissue. Skin and the cremaster muscle were sewed with surgical wire. After 5hrs the animals were sacrificed and both testes removed. The testes were snap frozen for RNA extraction and subsequent real time RT-PCR analysis. Based on the findings of an experiment employing a colored test substance to determine the distribution of injection solutions in the testis, only tissue around the injection site was used for gene expression analyses.

3.2.3 PAR2 activation in vivo using specific stable synthetic peptides

Because human tryptase, in addition to activating PAR2, causes a wide variety

of proteolytic effects (Sommerhoff, 2001, Brown et al., 2002), subsequent experiments utilized a specific PAR2 activating peptide; a peptide corresponding

to a PAR2-activating peptide with an N-terminal furoyl group modification,

2-furoyl-LIGRLO-NH2 was reported to be equally effectiveto and 10 to 25 times

more potent than SLIGRL-NH2 for increasing intracellular calcium in cultured

human and rat PAR2-expressing cells, respectively. PAR2-f-AP

([2-furoyl]-LI-GRLO-NH2 represents the most potent, metabolically stable and selective

activator of PAR2 in biological systems described to date (McGuire et al.,

2004a).

Rats (200g; n=5-10/group) received 50µl of PAR2-f-AP ([2-furoyl]-LIGRLO-NH2;

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al., 1998a) or a sham control (isotonic saline) under the testicular capsule as described above. To study the involvement of NO production and prostaglandin synthesis in PAR2-stimulated cytokine expression, two groups of rats received a

subcutaneous injection of either a NOS-inhibitor (L-NAME: L-nitro L-arginine methyl ester, 30mg/kg, Merck Biosciences GmbH, Schwallbach, Germany) or a COX-2 antagonist (Metacam®: Meloxicam; 0,3mg/kg; Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany) 20 min prior to the administration of [2-furoyl]-LIGRLO-NH2 or the control peptide. After 5hrs, animals were killed,

testes removed and one testis was used for RNA extraction and RT-PCR analysis and the other for histology as described above.

3.2.4 Myeloperoxydase (MPO) staining

MPO, an iron-containing protein, is found in the azurophilic granules of neutrophilic polymorphonuclear leukocytes (PMNs) and in the lysosomes of monocytes. MPO is most abundant in the granules of neutrophils. Monocytes contain only about a third of the MPO present in neutrophils.

We used a standard protocol (Hematology laboratory, Uniklinikum Giessen) for detection of MPO containing neutrophils. Testicular cryosections (12µm) from EAO and adjuvant control groups were fixed for 30 seconds in a solution containing ethanol:formalin (9:1, v:v) and immediately washed in tap water. Slides were further incubated for 15min in a “working solution” containing 5mg 3-amino-9-ethyl-carbazol (AEC, myeloperoxidase substrate), 3ml DMSO, 25ml 0.1M Michaelis’ buffer and 250µl 0.3% H2O2. After incubation, the slides were

washed for 5min in tap water and stained for 10 min with Mayers-Hemalaun solution. Finally the slides were washed 20 min in flowing tap water, air-dried, mounted in glycerin-gelatin and photographed using a trans-luminescence microscope (Carl Zeiss AG, Göttingen, Germany). MPO containing neutrophils have a red stained cytoplasm Mayers Hemalaun stains all nuclei blue.

3.2.5 Staining and quantification of mast cells

Cryosections of testis were cut at a thickness of 12µm. The slides were fixed in ice-cold isopropanol for 10 min and washed 3 times, for 5 min each in PBS. Toluidin blue O (Waldeck, Münster, Germany) stock solution (1% toluidin blue in 70% ethanol) was diluted 1:10 in freshly prepared 1% NaCl and added to the specimens. Metachromasia, tissue elements displaying a different color from

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the dye solution after staining, is dependent on the pH, dye concentration and temperature of the basic dye. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements. Slides were stained for 20sec or until metachromasia developed, washed in distilled water, dried on a heating plate (70°C) and mounted in LR white resin. For quantification, the slides were examined in a trans-luminescence microscope (Carl Zeiss AG, Göttingen, Germany) using the 20-fold objective. On each section both the metachromatically stained cells and the cross-sectioned seminiferous tubules were counted and results were expressed as a ratio mast cell/tubule cross section.

3.2.6 PAR2 immunohistochemistry on testicular paraffin sections

Paraffin sections (7µm) were dewaxed in xylene and rehydrated in decreasing concentrations of alcohol. Specimens were incubated in 3% H2O2 for 30min in

the dark to block endogenous peroxidases, followed by 10% normal donkey serum and 4% bovine serum albumin in PBS for 1hr to minimize unspecific protein-protein interactions, prior to being incubated overnight at 4°C with a goat anti-rat PAR2 (C-17, Santa Cruz, CA, USA). Source and dilution of

antibodies are presented in table 3.2.6 After washing in PBS/tween, the sections were incubated for an hour with a donkey anti-sheep/goat biotinylated secondary antibody followed by incubation with HRP-conjugated streptavidin for 30 min. The color reaction was developed with diaminobenzidine, the slides were coverslipped in aqueous mounting medium (Cristalmount, Sigma-Aldrich) and finally photographed.

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Table 3.2.6:

3.2.7 PAR2 immunohistochemistry on isolated peritubular cells

Peritubular cells were plated on glass coverslips in 6 well plates. Fixation was done in ice-cold isopropanol for 10 min. Unspecific protein interactions were blocked by incubation at room temperature for 1h with a blocking solution containing 10% normal donkey serum and 4% bovine serum albumin in PBS. The primary antibody goat anti-rat PAR2 antibody (C-17, Santa Cruz, CA, USA)

was diluted 1:25 in blocking solution and applied to slides overnight at 4°C. Than the slides were washed three times with PBS for at least 1h. The donkey anti-sheep/goat biotinylated antibody (Amersham, Buckinghamshire, UK; 1:400 in PBS) was incubated at room temperature for 1h. After a 1h washing step, slides were incubated for 1h in dark at room temperature with streptavidin Texas-Red conjugated 1:100 diluted in PBS. After a final washing step of 1h the slides were mounted in a DAPI containing mounting medium (Vector Laboratories, Burlingame, CA) and photographed using a fluorescence microscope (Carl Zeiss AG, Göttingen, Germany). Source and dilution of antibodies are presented in table 3.2.6.

3.2.8 Double immunofluorescence on isolated testicular macrophages

Testicular macrophages plated on glass coverslips in 24 well plates were fixed with ice-cold methanol for 10 min. Thereafter slides were washed 3 times with PBS. Unspecific protein interactions were blocked by incubation for 1h at room

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temperature with a blocking solution containing 10% normal donkey serum and 4% bovine serum albumin in PBS. The primary antibodies goat anti-rat PAR2

(C-17, Santa Cruz, CA, USA; 1:25), mouse anti rat CD68 (ED1, Serotec, Oxford, UK; 1:40) and mouse anti rat CD163 (ED2, Serotec, Oxford, UK; 1:40) were all diluted in blocking solution and applied together on the slides for 12-15h at 4°C. Than the slides were washed three times with PBS for at least 1h. The donkey anti-sheep/goat biotinylated antibody (Amersham, Buckinghamshire, UK; 1:400 in PBS) was applied for 1 h at room temperature. After a 1h washing step slides were incubated 1h in the dark at room temperature, with a pool of donkey anti mouse FITC (Dianova, Hamburg, Germany) 1:400 and streptavidin Texas-Red conjugated 1:100 diluted in PBS. After a final washing step of 1h the slides were mounted in a DAPI containing mounting medium (Vector Laboratories, Burlingame, CA) and photographed using a confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany). Source and dilution of antibodies are presented in table 3.2.6.

3.2.9 Double immunofluorescence on testicular cryosections

Cryosections of testis were cut at a thickness of 12µm. After drying for 20min at room temperature the slides were fixed in ice-cold methanol for 10 min. After that slides were washed 3 times with PBS. To minimize the unspecific protein binding the slides were incubated for 1h at room temperature with a blocking solution containing 10% normal donkey serum and 4% bovine serum albumin in PBS.

Thereafter specimens were treaded overnight at 4°C with pooled primary antibodies in the following combinations: 1) smooth muscle actin (SMA) and PAR2; 2) SMA and Ki67; 2) ED1/ED2 and Ki67. Corresponding fluorochrome

labeled secondary antibodies were co-applied in the dark for 1h at room temperature. Slides were mounted in a DAPI containing mounting medium (Vector Laboratories, Burlingame, CA) and photographed using a fluorescence microscope (Carl Zeiss AG, Göttingen, Germany). Source and dilution of antibodies are presented in table 3.2.6.

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3.2.10 Cell culture experiments

3.2.10.1 Isolation and culture of peritubular cells (PTs)

Peritubular cells (PTC) were prepared from 19-day-old rats according to a modified version of the protocol described by (Hoeben et al., 1995). Rats were killed with CO2 and testes were removed, rinsed once in 1% iodine alcohol, and

then washed several times in PBS (Dulbecco’s PBS without Ca2+ and Mg2+, PAA Laboratories, Cölbe, Germany). After decapsulation the tissue was minced in PBS followed by a 30 min incubation at 32°C in PBS containing 0.25% trypsin and 10µg/ml DNase I (Roche, Mannheim, Germany) under constant shaking. The enzymatic reaction was stopped by the addition of 5mg/ml trypsin inhibitor (Roche) in PBS and the tubule fragments were allowed to settle for 10-20 min. The supernatant was removed and the pellet was rinsed in 10-20ml PBS, then 2.5% trypsin inhibitor, followed by six to eight washes in 30ml PBS. Subsequently, the fragmented tubules were incubated for 10min at 32°C in PBS containing 1mg/ml collagenase, 1mg/ml hyaluronidase and 10µg DNase I (Roche) in a shaking water bath. Thereafter, 30ml of PBS were added and tubule fragments were allowed to settle for 10-20 min. The PTC containing supernatant was collected, supplemented with 20ml RPMI standard medium (PAA Laboratories) containing 10% fetal calf serum (FCS) and centrifuged at 500xg for 10min at room temperature. PTC from 20 immature animals were seeded into ten 75cm2 cell culture flasks and placed at 37°C in a humidified incubator in 5% CO2 atmosphere. For experiments, PTC were starved in a

FCS-free RPMI medium and moved to a 32°C/5%CO2 incubator one day prior

to stimulation.

3.2.10.2 Stimulation of peritubular cells with PAR

2

synthetic

agonists

As FCS contains factors of coagulation that could possibly activate PAR2 the

FCS concentration was gradually reduced before stimulating the cells with PAR2 agonists. Technically, FCS concentration was diminished in gradual

steps, by incubating the cells at 32°C every hour with medium containing decreasing FCS concentrations (5%, 2,5% and 1%).

PCs were stimulated after 24hrs of incubation in 1%FCS medium. Cells were treated with 10-4 M of either PAR2-activating peptide (SLIGRL-NH2, PAR2-AP)

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or the reverse peptide (LSIGRL-NH2, PAR2-RP) as control. Stimulation time

was 5 and 10 min for the analysis of phosphorylation of MAP kinases and 3, 6 and 9hrs for the assessment of cytokine expression using real time PCR.

3.2.10.3 Isolation and culture of testicular and peritoneal

macrophages

Testicular macrophages

For each experiment, four rat testes were decapsulated in 10ml ice-cold endotoxin free DMEM:F12 medium (Life Technologies, Karlsruhe, Germany). The seminiferous tubules were gently prized apart using curved forceps following the method described by (Hedger and Eddy, 1986). Enzymes were not used because these would activate the macrophages. Dissociated tubules were transferred in a new Falcon tube and the volume adjusted to 50ml. The tubule fragments were allowed to settle for 5min, and then the supernatant was transferred in a new clean tube, cells were counted without differentiating between cell types and centrifuged at 300g for 10min at 4°C to sediment the interstitial cells. The supernatant was removed and the pellet was resuspended in at 5*106 cells/ml in DMEM:F12. Cells were plated onto small Petri-dishes (50mm diameter) for RNA isolation (3ml each well) or in 24 well plates containing each a small glass coverslip (for further immunofluorescence experiments or for assessment of [Ca2+]i) (500µl each well) and incubated at

32°C for 30min. Contaminating cells were removed by washing the wells four times with DMEM:F12 taking advantage of the rapid adherence of macrophages to plastic surfaces.

Peritoneal macrophages

Two rats were killed with an overdose of Isofluoran and 50ml DMEM:F12 was injected in the peritoneal cavity. After gentle massage of the abdomen the medium was collected from the peritoneal cavity by peritoneal puncture and centrifuged at 300g for 10min to sediment the macrophages. Supernatant was removed and the pellet was resuspended in 1ml DMEM:F12. Cells were counted and the concentration adjusted to 5x106 cells/ml. Peritoneal

macrophages were plated in Petri dishes (50mm diameter) 1.8x106 cells/dish or in 24 well plates containing each a small glass coverslip (for further immunofluorescence experiments or for assessment of [Ca2+]i) and incubated at

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37°C for 30min in BSA free medium prior to vigorously wash the dishes 5 times with DMEM:F12 in order to remove non adherent cells. Outcome was generally approximately 50% of total plated cells. For RNA isolation, 5 similar preparations were polled after stimulation.

3.2.10.4 Stimulation of macrophages with PAR

2

synthetic

agonists

Cultured macrophages (peritoneal and testicular) were stimulated directly after the last washing step. Cells were treated with 10-4 M of either PAR2-activating

peptide (SLIGRL-NH2, PAR2-AP) or the reverse peptide (LSIGRL-NH2, PAR2

-RP) as control. As negative control cells received PBS and as positive control they received 2µM LPS in PBS. All chemicals used for stimulation were pipetted in the medium in volumes of 10-30µl of the corresponding stock solutions. After 6h stimulation time, cells were washed two times with sterile PBS and lysed with 200µl RLT buffer (Rneasy® Mini Kit, Qiagen, Hilden, Germany). The lysates were passed through a 24G needle 10 times, up and down using a sterile 1ml syringe and than stored at -80°C. Lysates from 6 experiments were pooled prior to RNA isolation as described at 3.2.10.3.

3.2.11 Assessment of intracellular free Ca2+ concentration

PTC and macrophages were grown as a monolayer on coverslips and incubated at 32°C for 30 min with 3 µM Fura-2-AM (1 mM stock in dimethylsulfoxide [DMSO]; Molecular Probes, Leiden, Netherlands). Extracellular free Fura-2-AM was removed by rinsing the cells three times with Hepes buffer salt solution (HBSS, pH 7.4). To allow complete hydrolysis of the intracellular Fura2-AM, the cells were incubated a further 30 min at 32°C with 5% CO2. After an additional washing step repeated twice, the coverslips were

placed into a HBSS containing measuring chamber (Delta T System, Olympus, Hamburg, Germany) and examined with a 40X water immersion objective and a 10X ocular in an Olympus microscope (Olympus BX50WI, Olympus, Hamburg, Germany). The cells were stimulated at 340 nm and 380 nm using a monochromator (T.I.L.L. Photonich GmbH, Martinsried, Germany). Emission intensities were recorded as a ratio (340nm/380nm) at 510 nm with a photomultiplier (T.I.L.L. Photonich GmbH, Martinsried, Germany). The

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background-corrected ratiometric signal R was determined by applying the standard equation:

[Ca2+] = Kd (R -Rmin)/(Rmax-R),

where Rmin and Rmax were derived from the experimental readings. All

measurements were performed between 60 and 90 minutes after addition of the dye. Data was aquired and analyzed using the T.I.L.L.Vision-v.4.0 software (T.I.L.L. Photonich GmbH, Martinsried, Germany). Cells were initially incubated with the reverse peptide (LSIGRL-NH2, PAR2-RP; 10-4 M) followed by addition

of 10-4 M PAR2-activating peptide (SLIGRL-NH2, PAR2-AP). Trypsin (100nM)

was used as natural PAR2 agonist and thapsigargin (10-7-10-5 M) as positive

control.

3.2.12 Western Blot analysis

PTCs were lysed in Laemmli buffer (5% bromophenol blue, 5% mercaptoethanol, 62.5 mM Tris-HCL, 20% glycerol, and 2% SDS). Samples were boiled, the proteins separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (HybondTM-ECLTM; Amersham Biosciences, Braunschweig, Germany) using the PerfectBlueTM semidry electroblotter (PeqLab Biotechnologie, Germany). To minimize non-specific protein binding, the membrane was blocked for 1hr in PBS containing 0.1% Tween-20 and 5% dry milk powder. The presence of phosphorylated p44/42 (ERK1/2) was detected using a mouse monoclonal antibody raised against phospho-p44/42 MAP kinase (thr202/Tyr204, see table 1), followed by horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:5000; Perbio Science, Bonn) and enhanced chemiluminescence detection (ECL, Amersham Biosciences). After exposure, the nitrocellulose membrane was stripped, blocked again under the same conditions and total p44/42 detected using a rabbit polyclonal antibody directed against p44/42 (see table 1) and a goat anti-rabbit horseradish peroxidase-coupled secondary antibody (1:3000; Perbio Science, Bonn, Germany) followed by ECL visualization.

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3.2.13 Working with RNA

Because of the ubiquitous presence RNases, working with RNA requires special care for assuring an RNase-free environment. The entire work with RNA samples involved the use of sterile autoclaved single-use instruments and hot-sterilized glassware, respectively. Alternatively, the instruments were treated for at least 30min with 3% H2O2 before using. At each experimental step single-use

gloves were worn.

3.2.14 Isolation of total RNA from cultured cells and tissues

Cell culture flasks containing maximum 107 peritubular cells were washed two times with PBS before adding 600µl RLT buffer (Rneasy® Mini Kit, Qiagen,

Hilden, Germany) containing 1%(v/v) ß-mercapto-ethanol. Cells were scraped from the surface of the flask before aspiring the cell content in a 1ml syringe through a 24G needle. The cell suspension in RLT buffer was discarded in a clean 1.5ml reaction tube and passed another 10 times through the same 24G needle. Samples were loaded on a QIAshredder spin column placed in a 2ml collection tube and centrifuged at maximum speed (13000rpm) for 2 min. The flow through was collected for each sample and used for RNA isolation with Rneasy Mini Kit (Qiagen, Hilden, Germany).

Tissue samples (max 30mg) were transferred each in 1.5ml reaction tubes containing 600µl RLT buffer with 1%(v/v) ß-mercapto-ethanol and one tungsten carbide bead (Qiagen, Hilden, Germany). Tissue samples were homogenized for 2.5min in a Retsch® MM300 (Retsch GmbH & Co, Hann, Germany) mixer mill at 30 agitations/s. Following the homogenization step, samples were centrifuged for 1 min at 10000rpm and the supernatant loaded on a QIAshredder spin column placed in a 2ml collection tube and centrifuged at maximum speed (13000rpm) for 2 min. The flow through was collected for each sample and used for RNA isolation using the Rneasy Mini Kit (Qiagen, Hilden, Germany).

After 6h stimulation time, the testicular and peritoneal macrophages were washed two times with sterile PBS and lysed with 200µl RLT buffer. The lysates were passed 10 times through a 24G needle, up and down using a sterile 1ml syringe and than loaded on a QIAshredder spin column placed in a 2ml collection tube and centrifuged for 2 min at maximum speed (13000rpm). The

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flow through was collected for each sample and stored at -80°C. Lysates from 6 experiments as described at 3.2.10.3 and 3.2.10.4 were pooled prior to RNA isolation.

To the cell or tissue lysates (obtained as described above) an equal volume (400-600µl) of 70% ethanol was added and mixed by pipetting. Samples were then loaded on RNeasy mini spin columns and centrifuged for 15s at 12,000rpm. The flow-through was discarded. The columns were washed by adding 700µl RW1 (Rneasy® Mini Ki) buffer and the flow-through was discarded. The columns were transferred in a new 2ml collection tube and washed 2 times with 500µl RPE buffer (Rneasy® Mini Kit). Each time the columns were centrifuged for 15s at 12,000rpm and the flow-through discarded. After the last washing step, the columns were centrifuged for 2min at 12000rpm to dry the silica membrane of the RNeasy mini spin column. The total RNA was diluted in 20-40µl of RNase-free water and the RNases inactivated by addition of 1% (v/v) RNase inhibitor (Promega, Manheim, Germany). RNA samples were stored at –80°C.

3.2.15 Assessment on RNA concentrations by spectrophotometric analysis

The spectrophotometer measures the light-absorption of different samples at defined wavelengths. The maximum absorption of diluted RNA is at λ = 260nm. Based on the Lambert-Beer law:

A = c*l*ε

the concentration (c) of a substance could be calculated if the absorption (A) measured through a well with the length (l) and the extinction coefficient (ε) are known.

The assessment of RNA concentration was performed using a spectrophotometer (Ultrospec2100pro, Pharmacia, Erlangen, Germany) at 260nm. At the same time the ratio OD260/OD280 was calculated and gave an

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OD260/OD280 values between 1.8 and 2.0. Values smaller than 1.8 are an

indication for protein contamination (Ibelgaufts et al., 1982). 3.2.16 DNase Digestion

For each sample 2.5µg RNA were taken separately in a new reaction tube together with DNase buffer and 3U DNase/µgRNA, (DNase I RNase-free, 10U/µl, Roche, Manheim, Germany) for each sample. The reactions were brought to an end volume by adding appropriate volumes of water (see reaction table below) and samples were incubated for 35min at 37°C. The DNase was inactivated at 72°C for 10 min. After that samples were transferred on ice for 2 min, centrifuged and stored at –80°C.

DNase digestion reaction table:

Volume

Component

Xµl RNA (corresponding to 2.5 µg)

0.9µl DNase I RNase free (10U/µl, Roche diagnostics, Manheim)

2.0µl 10X DNase I buffer

Up to 20µl RNase free water

20µl TOTAL

The efficiency of DNase digestion was assessed by PCR amplification of GAPDH housekeeping gene. Lack of GAPDH (glycerin-aldehyd-3-phosphate-dehydrogenase) gene amplification proved the complete digestion of genomic DNA. Samples were stored at -80°C.

3.2.17 Reverse transcription

After removing the genomic DNA, the total RNA samples were subject to reverse transcription (RT) into complementary DNA (cDNA).

The RT was performed (see reaction tables below) using Oligo(dT) (Promega) primers and the reverse transcriptase from mouse Moloney-Leukemia Virus (M-MLV, Promega).

DNase digested RNA samples (2.5µg in 20µl, see DNase reaction table) were mixed with 2µl Oligo dT (12-18) primer and denaturated for 10min at 70°C. After

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